Muscle SP cells are a heterogeneous population of primitive cells that demonstrated hematopoietic and myogenic differentiation potential in vivo. First discovered in mice, these cells hold promise for optimization of cell-based therapy for muscular dystrophy. To date, little is known about human-derived muscle SP cells and their presumed clinical potential. The goals of this proposal are to study the differentiation potential of human fetal and adult-derived muscle SP cells, and to optimize their use in pre-clinical experiments using newly generated muscular dystrophy mouse models. These goals will be achieved via the following specific aims: Aim 1.1. Study the potential of unfractionated human muscle-derived SP and MP cells to differentiate into myogenic or hematopoietic cells in vitro. Human samples will be obtained from autopsy fetal and adult tissues and from adult-derived discarded surgical tissue. Aim 1.2. Fractionate human fetal and adult muscle SP and MP cells based on the expression of the cell surface antigens CD34, CD90 and CD133. Compare the ability of fractionated versus unfractionated cells to differentiate into myogenic or hematopoietic cells in vitro. Aim 2.1. Compare the ability of unfractionated and fractionated muscle-derived SP and MP cells to fuse into dystrophic myofibers in vivo. Perform intramuscular injections into NOD/RAG1/null-DMD/mdx5cv mice using the fetal and adult-derived human muscle SP and MP cell fractions enriched for myogenic precursors (Aim 1.2). Aim 2.2. Assess the clinical potential of human fetal and adult-derived muscle SP or MP cells with myogenic activity to engraft dystrophic skeletal muscles in vivo. Perform intra-arterial and intravenous injections of human muscle SP or MP cells in non-irradiated NOD/RAG1/null-DMD/mdx5cv. Aim 2.3. Assess the ability of fractionated human fetal and adult-derived muscle SP or MP cells to differentiate into hematopoietic cells in vivo. Inject unfractionated and fractionated cells with hematopoietic activity (Aims 1.1 and 1.2) into the circulation of lethally irradiated NOD/RAG1/null/mdx5cv or NOD/RAG1/null-Pfp/null DMD/mdx5cv mice. These studies will enhance our basic knowledge on human muscle SP cells, their differentiation potential in fetal and adult tissue, and will evaluate their therapeutic promise for muscular dystrophy.